Does low intracellular pH stop the motion of the Bulla circadian pacemaker?

The eye of the mollusk Bulla has proven itself useful as an in vitro neural circadian pacemaker. Here, we report that treatments applied to lower intracellular pH may stop the motion of this circadian pacemaker in a phase-dependent manner. Lowering the extracellular pH of the artificial seawater bath to 6.9, or application of the stilbene derivatives 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) or 4,4-di-isothiocyanostilbene-2,2′-disulfonic acid (DIDS), abolishes the circadian rhythm in optic nerve compound action-potential frequency. Because these treatments are known to lower intracellular pH, these data suggest that the pacemaker may be inhibited by low intracellular pH. In order to assess the state of the pacemaker during low extracellular pH treatment, pulses of seawater at pH 6.8 were applied, and the phase of the rhythm subsequent to the pulse was observed. All pulses started 1 hr after subjective dusk [circadian time (CT) 13] and were applied to eyes in constant darkness; pulse lengths varied from 4 to 47 hr for different preparations. The phases of the eye rhythms following pulses that ended before subjective dawn (about CT 24) were not different from untreated preparations. However, for pulses longer than 11 hr and therefore ending after subjective dawn, the subsequent phase of the rhythm was a function of the ending time of the pulse. These data suggest that the pacemaker's motion was stopped at dawn during the low-pH treatment and resumed following restoration of normal pH. To distinguish between phase and duration dependence of this effect in the above experiment, phase shifts were obtained to 14- hr pulses of pH 6.8 seawater applied at three different phases.